WO2007140962A2 - Improved measurement of vitamin d - Google Patents

Improved measurement of vitamin d Download PDF

Info

Publication number
WO2007140962A2
WO2007140962A2 PCT/EP2007/004924 EP2007004924W WO2007140962A2 WO 2007140962 A2 WO2007140962 A2 WO 2007140962A2 EP 2007004924 W EP2007004924 W EP 2007004924W WO 2007140962 A2 WO2007140962 A2 WO 2007140962A2
Authority
WO
WIPO (PCT)
Prior art keywords
vitamin
metabolite
sample
binding protein
releasing reagent
Prior art date
Application number
PCT/EP2007/004924
Other languages
English (en)
French (fr)
Other versions
WO2007140962A3 (en
Inventor
Uwe Kobold
Thomas Duelffer
Michael Grol
Rupert Herrmann
Herbert Von Der Eltz
Leopold Von Proff
Original Assignee
Roche Diagnostics Gmbh
F. Hoffmann-La Roche Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roche Diagnostics Gmbh, F. Hoffmann-La Roche Ag filed Critical Roche Diagnostics Gmbh
Priority to AT07725793T priority Critical patent/ATE452343T1/de
Priority to CA2649338A priority patent/CA2649338C/en
Priority to EP07725793A priority patent/EP2030026B1/de
Priority to JP2009513585A priority patent/JP4953477B2/ja
Priority to DE602007003833T priority patent/DE602007003833D1/de
Priority to CN200780021252.0A priority patent/CN101467048B/zh
Publication of WO2007140962A2 publication Critical patent/WO2007140962A2/en
Publication of WO2007140962A3 publication Critical patent/WO2007140962A3/en
Priority to US12/326,972 priority patent/US8003400B2/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/20—Oxygen containing
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/20—Oxygen containing
    • Y10T436/203332—Hydroxyl containing

Definitions

  • the present invention relates to a method of measuring a vitamin D metabolite in a sample, the method comprising the steps of (a) treating said sample with a vitamin
  • the present invention also relates to methods for determining the vitamin D status of a subject, for use in the diagnosis of disease, and to agents and kits for use in performing the methods of the invention.
  • vitamin D status The level of circulating vitamin D or vitamin D metabolites in a subject is referred to as vitamin D status.
  • Malnutrition with regard to vitamin D is an important factor in the cause of a number of diseases including rickets in children and osteomalacia and may be even osteoporosis in adults.
  • Knowledge of the vitamin D status by measurement of Vitamin D and its metabolites in a clinical sample is very helpful in the assessment of a patient and may by of help to the clinician in establishing a diagnosis.
  • vitamin D is available in two forms, i.e., either as vitamin D 2 or as vitamin D 3 .
  • Vitamin D 2 is produced outside the body by irradiation of ergosterol from yeast and fungi, and is found in a human being when taken up in the form of fortified foods or pharmaceutical preparations.
  • Vitamin D 3 is formed in animals from 7-dehydrocholesterol upon exposure to ultraviolet light.
  • Vitamin D 3 is also available in the diet, for example from fish liver oils.
  • Nutritional vitamin D in the form of vitamin D 2 or D 3 , after its up-take into the human body is rapidly converted to the circulating metabolite, 25-hydroxyvitamin D, which is found outside cells, tightly bound to circulating vitamin D binding protein. Due to the rapid conversion of vitamin D to its first metabolite, 25-hydroxyvitamin D, measurement of vitamin D does not give a useful indication of the vitamin D status of a subject.
  • Other metabolites of vitamin D such as l ⁇ ,25-dihydroxyvitamin D, circulate at a concentration 1000 times lower than non-l ⁇ -hydroxylated metabolites such as 25-hydroxyvitamin D, and so do not contribute significantly to the estimation of total circulating vitamin D metabolite.
  • 25-hydroxyvitamin D is the metabolite with the highest serum concentration, and is easy to measure. It has therefore become the most common marker of vitamin D status in a subject.
  • Vitamin D metabolites also bind to other serum proteins, e.g. to albumin, however, much less tightly than to the binding protein. It is generally accepted that methods facilitating the release of vitamin D out of a vitamin D-vitamin D binding protein complex, will also be appropriate to set it free out of the other less strong complexes as well. The rapid and strong binding of various vitamin D metabolites to vitamin
  • vitamin D binding protein thus is the major concern in detection of a vitamin D metabolite and enormously hampers the measurement. All presently known methods require that vitamin D metabolites have to be released from vitamin D binding protein. In such procedures vitamin D binding protein usually is denatured. This typically also requires an extraction step which separates vitamin D binding protein along with other denatured proteins from the vitamin D metabolite of interest and removes the denatured protein fraction from the sample. This way the vitamin D metabolites of interest become available in a separate fraction and is more easily handled and detected.
  • Extraction has been achieved by a number of methods, including solvent based extraction by adding to the sample an organic solvent such as chloroform, hexane or ethyl acetate and hexane. The organic and aqueous layers are separated and the solvent evaporated. The residue is then reconstituted in a water miscible solvent such as ethanol. Reverse phase cartridge extraction methods have also been used. Other traditional methods include the use of HPLC and mass spectroscopy to achieve separation of individual vitamin D metabolites and exclude from the sample interfering factors such as binding proteins.
  • Armbruster, F.P., et al. teach that a serum or plasma sample may be treated by ethanol in order to release a vitamin D metabolite out of its complex with vitamin D binding protein.
  • the precipitated protein is spun down and a vitamin D metabolite is obtained in the supernatant.
  • the vitamin D metabolite comprised in such supernatant can be easily detected e.g. by any liquid chromatography based method.
  • Holick, M.F. and Ray, R. (U.S. Pat. No. 5,981,779) describe methods for assaying vitamin D and its metabolites. Their procedure is based on a competitive binding assay using a purified vitamin D binding protein as the specific binding agent. A prerequisite for this assay also is that a vitamin D metabolite of interest has first to be isolated from the sample, separated from its binding protein and only thereafter may be measured.
  • steroid analogues are used to displace these hormones from their binding proteins. These steroid analogues must bind to the relevant steroid binding proteins, and at the same time must not cross react with the antibody used in the immunoassay.
  • the steroid analogue saturates the steroid binding protein, displacing the steroid and allowing the steroid to bind to the antibodies of the immunoassay.
  • the present invention helps to overcome or to at least ameliorate some of the problems associated with the prior art procedures, by providing a method for measuring a vitamin D metabolite present in a sample, whereby the improved method is easily combined and based on a standard liquid chromatography procedure, and does not require any manual handling.
  • the present invention relates to a method of measuring a vitamin D metabolite in a sample, the method comprising the steps of (a) treating said sample with a vitamin D releasing reagent under conditions appropriate to release a vitamin D metabolite from vitamin D-binding protein and not to cause protein precipitation, (b) subjecting the treated sample obtained in step (a) to a chromatographic separation, and (c) measuring a vitamin D metabolite during or after said chromatographic separation.
  • the present invention satisfies the pressing need for a simple yet effective method for measuring a vitamin D metabolite of interest in a serum or plasma sample. It is based upon the surprising discovery of an appropriate vitamin D releasing reagent that enables release and direct online chromatographic separation of a vitamin D metabolite from vitamin D binding protein. This way the amount of a vitamin D metabolite can be detected or measured, without requiring its extraction from the sample. In essence, the invention discloses for the first time that appropriate vitamin D releasing reagents may eliminate the need of an extraction step in the online detection of vitamin D. In addition a method based on the use of the novel and appropriate vitamin D releasing reagent is suited to routine use in clinical biochemical laboratories.
  • the present invention may be performed on any sample of plasma or serum, preferably from an individual.
  • the individual whose plasma or serum is to be analyzed may be one for whom it is desirable to determine vitamin D status.
  • Measuring a vitamin D metabolite of interest present in a sample of plasma or serum may include both qualitative as well as quantitative measurements, i.e. detecting the presence of a vitamin D metabolite of interest in the sample, or determining the amount of a vitamin D metabolite present, respectively.
  • the amount of a vitamin D metabolite of interest is compared with a key detailing whether the amount measured represents a deficiency or an excess of said vitamin D metabolite.
  • any one or more metabolites of vitamin D may be measured in the method of the present invention.
  • a specific vitamin D metabolite of interest is measured in a sample, although it is envisaged that for some applications it may be preferred to measure two or more types of the vitamin D metabolites in a sample.
  • the vitamin D metabolite of interest is selected from the group consisting of 25-hydroxyvitamin D 2 , 25-hydroxyvitamin D 3 , 24,25- dihydroxyvitamin D 3 , 25,26-dihydroxyvitamin D, and 1,25 dihydroxyvitamins D 2 and D 3 .
  • 25-hydroxyvitamins D 2 or D 3 are preferred vitamin D metabolites to be measured in the method of the invention.
  • the vitamin D metabolite is 25OH vitamin D 3 .
  • vitamin D By release of vitamin D is meant the full or partial separation of some or all of the vitamin D metabolites from vitamin D binding protein. It is preferred that substantially all of the vitamin D metabolites present in the sample are released from vitamin D binding protein.
  • the appropriate minimal concentration of a vitamin D releasing reagent is determined as the minimal concentration that results in the release of at least 99% of the bound vitamin D binding protein. These conditions mimic nicely the conditions found if vitamin D is released from its binding protein in a serum or plasma sample.
  • the minimal concentration of a candidate vitamin D releasing reagent as determined in the Biacore system is the same as the minimal concentration required for the efficient release of 25OH vitamin D 3 from vitamin D binding protein in a sample using that candidate vitamin D releasing reagent.
  • the vitamin D binding protein in the Biacore analysis is diluted in the candidate vitamin D releasing reagent.
  • the minimal final concentration of the vitamin D releasing reagent in a mixture with the sample under investigation has to be at least the same concentration as the one determined by Biacore analysis. If e.g. sample and vitamin D releasing reagent are mixed 1:1, the releasing reagent has to be double concentrated as compared to the Biacore setting before it is mixed with the sample. This way the minimal concentration determined as described above is present in the mixture of sample and releasing reagent.
  • vitamin D releasing reagent capable of achieving displacement, or separation, of the vitamin D metabolite 25-hydroxyvitamin D 3 from vitamin D binding protein and not resulting in protein precipitation may be used in step (a) of the method of the invention.
  • Preferred agents for use in the present invention are chemical reagents which may act by disrupting or destroying the bond between a vitamin D metabolite and vitamin D binding.
  • the vitamin D releasing reagent is based on a salt with a cation having a quaternary nitrogen-based ion. Also preferred the releasing reagent is based on a salt having a quarternary N-heterocycle as a cation.
  • Preferred cations are selected from the group consisting of pyrazolium cations, imidazolium cations, triazolium cations, pyridinium cations, pyridazinium cations, pyrimidinium cations, pyrazinium cations and triazinium cations.
  • Preferred cations are those based on an imidazolium heterocyclic nucleus.
  • the anion is selected from halogenated inorganic anions, nitrates, sulphates, carbonates, sulphonates and carboxylates.
  • the anion may be selected from chloride, hexafluorophosphate, tetrafluoroborate, trifluoroacetate, benzoate, salicylate, and rhodanide. Combinations of the above cations and anions in most cases are extremely good miscible with water. Many are even soluble without water.
  • Appropriate reagents for release of vitamin D are preferably selected from the group consisting of l-Butyl-4-methylpyridinium tetrafluoroborate; l-Butyl-3-methyl- imidazolium tetrafluoroborate; l-Butyl-3-methyl-imidazoliumoctylsulfate; 1- Butyl-3-methyl pyridiniumchloride; 1-Hexylpyridiniumchloride; 1 -Methyl- 1-octyl pyrrolidiniumchloride; N-Octylpyridiniumchloride; 3-Carbamoyl-l- octyloxymethyl pyridiniumchloride; KBr; KJ; and KSCN, and of combinations thereof.
  • such combination comprises five or less of these compounds.
  • a mixture of four, three or two of these compounds can be used.
  • the use of a single compound is preferred.
  • the reagent for differential hemolysis may also be selected from the group consisting of l-Butyl-4-methylpyridinium tetrafluoroborate; l-Butyl-3-methyl- imidazolium tetrafluoroborate; l-Butyl-3-methyl-imidazoliumoctylsulfate; 1-
  • Protein precipitation in the sense of the present invention is assessed by applying in a standardized manner a sample of plasma or serum treated with a candidate vitamin D releasing reagent to a standard frit e.g. to a frit as part of an HPLC column.
  • a candidate vitamin D releasing reagent does not cause precipitation, i.e. is appropriate for latter online LC
  • said reagent is mixed 1 to 1 with a sample of plasma or serum and incubated for at least 15 min and for at most 60 min at 2O 0 C. 50 aliquots of 10 ⁇ L of the thus processed sample are applied to a frit with a diameter of 2 mm and 0.5 ⁇ m pore size. The back-pressure is monitored. A candidate reagent for release of vitamin D that would cause an increase in backpressure of 20 bar or more - if the back-pressure for injection 50 and the backpressure for the first injection are compared to each other - would be deemed not to be appropriate.
  • the filter used in the above analysis is an HPLC frit.
  • such frit is made of stainless steel and is 1/32 inch thick.
  • the frit is part of an HPLC column of 20 mm in length, having an inner column diameter of 2 mm that is filled with 3.5 ⁇ m Symmetry® Cl 8 particles with a pore size of 100A° as bed material.
  • serum or plasma sample used for such assessment is obtained from a healthy individual, i.e. an individual having no known disease and biochemical values in the normal range.
  • the appropriate vitamin D releasing reagent is further characterized in that the (minimal) concentration required for release of vitamin D from vitamin D binding protein and the (maximal) concentration tolerated and not causing precipitation are at least two-fold apart. The broader the window between minimal and maximal concentration the more easy such reagent can be used in clinical diagnostic routine. It is further preferred that the vitamin D releasing reagent is used at a final concentration corresponding to the mean value plus/minus 25% of the minimal concentration and the maximal concentration. Further preferred the final concentration will be adjusted to be within plus or minus 20% of the mean value of minimal and maximal concentration.
  • the prior art reagents like ethanol or acetonitrile all cause precipitation if used in a high concentration
  • several of the now investigated reagents can be used in very high concentrations without causing protein precipitation at all.
  • the vitamin D releasing reagent of the present invention is used at a concentration of no more than 75% weight/volume, also preferred at no more than 50% weight/volume.
  • a plasma or serum sample treated with an appropriate vitamin D releasing reagent according to the present invention can be directly subjected to liquid chromatography.
  • Liquid chromatography is an extremely important analytical technique which is used for the separation, identification and quantitation of an analyte of interest, e. g. of a vitamin D metabolite.
  • an analyte of interest e. g. of a vitamin D metabolite.
  • the chemical components in a mixture are carried through a stationary phase by the flow of a liquid mobile phase. Separation in liquid chromatography is achieved by means of differences in the interactions of the analytes with both the mobile and stationary phases.
  • a stationary phase and a mobile phase appropriate to the analytes under investigation have to be chosen.
  • the user will identify chromatographic conditions appropriate to maintain the sharpness of analyte bands or peaks as a sample moves through the stationary phase column to the detector.
  • High Performance Liquid Chromatography also known as High Pressure Liquid
  • HPLC Chromatography
  • the mobile phase is chosen to ensure solubility of the sample solutes.
  • the stationary phase preferably micro particulate silica (bare or chemically modified) is used, because its high surface area accentuates the differences in solute-stationary phase interactions.
  • the use of a stationary phase that interacts strongly with solutes relative to solute mobile-phase interactions will result in very long retention times, a situation which is not analytically useful.
  • the stationary phase must be selected so as to provide weak to moderate solute interactions relative to those in the mobile phase.
  • the nature of the solute governs the type of LC selected. The stronger interactions should occur in the mobile phase to ensure sample solubility and ready elution, while the stationary phase should be responsive to more subtle differences among the solutes.
  • polar neutral compounds are usually better analyzed using a polar mobile phase together with a nonpolar stationary phase that distinguishes subtle differences in the dispersive character of the solutes.
  • HPLC high-density lipoprotein
  • Modifiers can be added to the mobile phase to control retention.
  • pH is an important variable in aqueous mobile phases.
  • Reversed-phase chromatography calls for the use of a non-polar stationary phase and a polar mobile phase (composed of one or more of the polar solvents, e.g. water, methanol, acetonitrile, and tetrahydrofuran).
  • a non-polar stationary phase and a polar mobile phase (composed of one or more of the polar solvents, e.g. water, methanol, acetonitrile, and tetrahydrofuran).
  • Ion-exchange chromatography involves ionic interactions.
  • the mobile phase must support ionization to ensure solubility of ionic solutes.
  • the stationary phase must also be partially ionic to promote some retention.
  • Size-Exclusion chromatography involves separations based on molecular size alone and ideally requires that there be no energetic interaction of the solutes with the stationary phase.
  • Affinity chromatography is based on a specific interaction, e.g. between the members of a specific binding pair, like antigen and corresponding antibody or receptor and corresponding ligand.
  • a first partner of a binding pair is bound to an appropriate stationary phase and used to capture the second partner of the binding pair.
  • the second partner can be released and isolated by appropriate means.
  • the so-called bed material e.g. silica particles in an RP-HPLC-application
  • the frit material usually is selected to have e.g. a smaller pore size as compared to the interparticle pore size of the bed material.
  • the diameter of the stationary phase particles usually is in the range of 1 to 10 ⁇ m. These small particles necessitate the high pressure used in HPLC.
  • the bed material usually is protected by a frit.
  • Typical frits have a pore size of 1 ⁇ m, 0.45 ⁇ m or 0.2 ⁇ m. The smaller the particles the smaller is usually the pore size of the frit. If a sample comprises a constituent capable of blocking an HPLC frit this is detrimental for any routine analysis. As the skilled artisan will appreciate blocking of the frit used in an HPLC column will occur the more rapidly the smaller the pore size of the frit and the smaller the column and correspondingly the frit diameter. In case the frit would not be selected appropriately, i.e. a too large pore size, the particle size of the column material would also matter and the column itself would block more rapidly the smaller the particles are. However, the skilled artisan will select the pore size of the frit to meet the requirements for protecting the column bed material.
  • a plasma or serum sample is e.g. treated with acetonitrile to release vitamin D from its complex with vitamin D binding protein a lot of proteins are denatured and precipitate.
  • Such sample can not be applied to an HPLC column in any routine setting, because it would block the column and cause a system shut down.
  • this HPLC step is performed online with the sample obtained by treatment with the vitamin D releasing reagent.
  • the stationary phase particles used in such HPLC step are in the range of 1 to 10 ⁇ m, also preferred in the range of 2 to 7 ⁇ m in diameter.
  • the frit used in such HPLC step has a pore size of 0.5 ⁇ m or also preferred of 0.2 ⁇ m.
  • vitamin D releasing reagent does not cause protein precipitation.
  • the analyte of interest can be detected by any appropriate means.
  • Appropriate and preferred detectors sense the presence of a compound passing through, and provide an electronic signal to a recorder or computer data station.
  • the output is usually in the form of a chromatogram and a substance of interest is usually found in a certain peak.
  • the peak area or the hight of the peak can be used to quantify the amount of analyte present in the sample investigated.
  • the detector for an HPLC system is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram. It is positioned immediately posterior to the stationary phase in order to detect the compounds as they elute from the column.
  • the detection and sensitivity parameters may be controlled by the skilled artisan.
  • detectors There are many types of detectors that can be used with HPLC. Some of the more common detectors include: Refractive Index (RI), Ultra-Violet (UV), Fluorescent, Radiochemical,
  • Refractive Index detectors measure the ability of sample molecules to bend or refract light. This property for each molecule or compound is called its refractive index. For most RI detectors, light proceeds through a bi-modular flow-cell to a photodetector. One channel of the flow-cell directs the mobile phase passing through the column while the other directs only the mobile phase. Detection occurs when the light is bent due to samples eluting from the column, and this is read as a disparity between the two channels.
  • Fluorescent detectors measure the ability of a compound to absorb and then re- emit light at given wavelengths, respectively. Each compound able to emit the fluorescence light has a characteristic excitation and emission wavelength. The excitation light passes through the flow-cell while the photodetector in orthogonal position measures the emited light at specific wavelength.
  • Radiochemical detection involves the use of radiolabeled material, usually tritium (3H) or carbon-14 (14C). It operates by detection of fluorescence associated with beta-particle ionization, and it is most popular in metabolite research.
  • Electrochemical detectors measure compounds that undergo oxidation or reduction reactions. This is usually accomplished by measuring gain or loss of electrons from migrating samples as they pass between electrodes at a given difference in electrical potential.
  • Mass spectrometry is an analytical technique used to measure the mass-to-charge ratio (m/z (or m/q)) of ions. It is most generally used to analyze the composition of a physical sample by generating a mass spectrum representing the masses of sample components.
  • the technique has several applications, including: identifying unknown compounds by the mass of the compound and/or fragments thereof; determining the isotopic composition of one or more elements in a compound; determining the structure of compounds by observing the fragmentation of the compound; quantitating the amount of a compound in a sample using carefully designed methods (mass spectrometry is not inherently quantitative); studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in vacuum); determining other physical, chemical or even biological properties of compounds with a variety of other approaches.
  • a mass spectrometer is a device used for mass spectrometry, and produces a mass spectrum of a sample to analyze its composition. This is normally achieved by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux.
  • a typical mass spectrometer comprises three parts: an ion source, a mass analyzer, and a detector.
  • Electron ionization and chemical ionization are used for gases and vapors.
  • chemical ionization sources the analyte is ionized by chemical ion-molecule reactions during collisions in the source.
  • Two techniques often used with liquid and solid biological samples include electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).
  • Other techniques include fast atom bombardment (FAB), thermospray, atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS) and thermal ionisation.
  • Nuclear magnetic resonance (NMR) detection is based on the fact that certain nuclei with odd-numbered masses, including H and 13 C, spin about an axis in a random fashion. However, when placed in a strong magnetic field, the spins are aligned either parallel or anti-parallel to the magnetic field, with the parallel orientation favored since it is slightly lower in energy. That magnetic nuclei can absorb RF energy when placed in a magnetic field of a specific strength. When this absorption occurs, the nucleus is said to be on resonance. Interestingly for analytical scientists, different atoms within a molecule resonate at different frequencies at a given field strength. The observation of the resonance frequencies of a molecule allows a user to discover structural information about the molecule.
  • Nephelometry is defined as the measurement of intensity of scattered light emanated from an illuminated volume of a suspension. The ratio of scattered intensity to illuminating intensity is compared with a standard of known properties.
  • Turbidometry is defined as the measure of the reduction of light transmitted due to particles in solution. It measures the light scatter as a decrease in the light that is transmitted through the particulate solution. Therefore, it quantifies the residual light transmitted.
  • Near-infrared detectors operate by scanning compounds in a spectrum from 700 to 1100 nm. Stretching and bending vibrations of particular chemical bonds in each molecule are detected at certain wavelengths. A vitamin D metabolite is preferably detected by mass spectroscopy.
  • the method according to the present invention is used for determining the vitamin D status of a subject.
  • kits comprising a vitamin D releasing reagent according to the present invention.
  • the kit preferably also comprises a key showing the correlation between the results of the assay with the amount of vitamin D metabolite present in the sample.
  • the kit preferably will also comprise instructions for use.
  • a big advantage of the method according to the present invention is that in case a diagnostic need exists to assess more than one vitamin D metabolite, this can easily be accomplished.
  • the sample is analyzed for at least two vitamin D metabolites of interest that are selected from the group consisting of 25- hydroxyvitamin D 2 , 1,25-dihydroxyvitamin D 3 , 25-hydroxyvitamin D 3 , 24,25- dihydroxyvitamin D 3 , 25,26-dihydroxyvitamin D 3 .
  • OH vitamin D 3 Preferably 25 OH vitamin D 3 , 1,25-dihydroxyvitamin D 3 and 24,25 dihydroxyvitamin D 3 are assessed in one run using the method according to the present invention.
  • the method according to the present invention can be combined with the advantages of using an isotope-labeled internal standard.
  • the present invention relates to a method of measuring a vitamin D metabolite in a sample, the method comprising the steps of a) adding an isotope labeled vitamin D metabolite to said sample, b) treating said sample with a releasing reagent under conditions appropriate to release the vitamin D metabolite from vitamin D-binding protein, c) subjecting the treated sample obtained in step (b) to liquid chromatography, and d) measuring the vitamin D metabolite during or after liquid chromatography, preferably by mass spectroscopy.
  • the present invention relates to a vitamin D releasing reagent appropriate to release 25 OH-vitamin D from vitamin D binding protein and not to cause protein precipitation, which additionally comprises an isotope-labeled vitamin D metabolite.
  • Said isotope-labeled vitamin D metabolite preferably is an isotope-labeled 25 OH-vitamin D 3 .
  • the concentration of the isotope-labeled vitamin D metabolite is known and preferably adjusted to match the physiologically relevant concentration of the vitamin D metabolite of interest.
  • the presnt invention relates to a kit comprising a vitamin D releasing reagent and in addition an isotope-labeled vitamin D metabolite, wherein said isotope-labeled vitamin D metabolite may be present as a separate component or is already contained within the vitamin D releasing reagent and wherein said releasing reagent is based on a salt having a quaternary N- heterocycle as a cation.
  • FIG. 1 Synthesis of biotinylated 25hydroxyvitamin D 3 The steps used in the synthesis of a biotin-25hydroxyvitamin D 3 - conjugate are depicted schematically.
  • Figure 2 Separation of vitamin D binding protein from 25hydroxyvitamin
  • valve settings of the automated HPLC system for sample injection mode and wash mode are given.
  • Figure 4 Analyte transfer The valve settings of the automated HPLC system for transfer of the analyte containing fraction from the extraction column to the analytical column are shown.
  • Figure 5 Analyte elution
  • valve settings of the automated HPLC system for isocratic analyte elution from the analytic column are given.
  • 25hydroxyvitamin D 3 is chemically activated at position 3 of the vitamin D scheme depicted in Formula I.
  • position 25 of Formula I is carrying an OH-group.
  • step 1.1 The nitrile obtained in step 1.1 is dissolved in 15 ml diethylether. Under stirring a suspension consisting of 7.5 mg lithiumhydrid in 7.5 ml diethylether is added. The mixture is stirred for one hour at room temperature (RT). Thereafter a suspension of 38.4 mg lithiumaluminumhydrid in 6.6 ml diethylether is added. The reaction mixture turns turbid and is stirred for a further hour at RT. Thereafter the reaction mixture is cooled to 0-5 0 C in an ice bath and slowly diluted by adding 35 ml of water in total. By adding 6.6 ml of a 10 M KOH the pH-value turns highly alkaline.
  • the organic material is extracted thrice with 65 ml methyl-tert.-butylether, each.
  • the pooled organic phase is dried by adding 5 g of water free sodium sulfate, filtered through a G3 frit and finally the organic solvent is removed by applying a vacuum. The remaining viscous solid is further dried by applying high-vacuum.
  • the raw material obtained in this step is dissolved in a mixture of 5 ml DMSO and
  • acetonitrile 3.0 rnL acetonitrile and purified via preparative HPLC.
  • the gradient applied goes within 100 min from 50% eluent B to 100% eluent B.
  • the column material is Vydac C18/3O ⁇ A/15-2O ⁇ m and the column has a diameter of 5 cm and a length of 25 cm. Chromatography is performed at RT with a flow rate of 30 ml/min. The elution is monitored at 226 nm.
  • the fractions comprising the desired product in a purity of at least 85% as determined by analytical HPLC are pooled and lyophilized.
  • the yield of the desired color free product is about 70%.
  • the Biacore® system is used to assess whether a reagent considered to be a potential candidate for releasing vitamin D from vitamin D binding protein will be efficient in releasing of vitamin D binding protein to a vitamin D metabolite.
  • a sensor chip coated with streptavidin (Sensor Chip SA, Biacore AB, BR-1000-32) is used for immobilization of a biotinylated vitamin D metabolite of interest.
  • the assessment is best performed by use of the vitamin D metabolite 25OH vitamin D 3 .
  • the sensor chip is first incubated with a saturating concentration of biotinylated 25hydroxyvitamin D 3 . Then the chip is loaded with a saturating amount of vitamin D binding protein. Then the vitamin D binding protein saturated chip is incubated with a sodium chloride solution one the one hand and a candidate vitamin D releasing reagent in various concentrations on the other hand. The release of vitamin D binding protein is monitored for 3 min.
  • a candidate vitamin D releasing reagent causing the release of at least 99% vitamin D binding protein in the above system is appropriate to meet the minimal requirements for a vitamin D releasing reagent used in the detection of a vitamin D metabolite in a sample of serum or plasma 25hydroxyvitamin D 3 .
  • the 25hydroxyvitamin D 3 -coated SA-chip is regenerated by washing it with 10 mM Gly/HCl pH 1.7 for 1 min.
  • a reference flow cell coated with biotin on the same chip was used as non-specific control. The data of the reference flow cell are subtracted from those of the biospecific flow cell. Thus specific data free of non-specific effects are obtained.
  • Stable isotope-labeled 25-hydroxyvitamin D 3 is used for internal standardization. Acetonitrile is added to the sample in order to release the analyte from vitamin D binding protein. Manual protein precipitation is performed, followed by online automated solid phase extraction with direct transfer to the tandem mass spectrometry system. Atmospheric pressure chemical ionization (APCI) in the positive mode is used. For native 25-hydroxyvitamin D 3 , the transition 401>257 m/z is recorded. For the internal standard labeled with six deuterium atoms, the transition 407>263 is recorded.
  • APCI Atmospheric pressure chemical ionization
  • 25-hydroxyvitamin D 3 (25-hydroxycholecalciferol) is procured from Sigma (Deisenhofen, Germany) (purity 98%; molecular weight 400.7).
  • a stock solution with a concentration of 3250 nmol/L is prepared in methanol.
  • Thermo Electron with APCI ion source Thermo Electron with APCI ion source.
  • LiChrospher RP- 18 ADS, 25 ⁇ m, 25 x 4 mm extraction column (Merck) is used in combination with a Rheodyne six-port high- pressure switching valve
  • the automated solid phase extraction procedure consisted of five steps:
  • the parameters of the atmospheric pressure ion source (APCI) and the mass spectrometer tuning parameters are set and optimized according manufacturer instructions in order to obtain maximum sensitivity for HVD detection.
  • MS analyser resolution is set to a peak width of 0.7 amu.
  • Argon is used as collison gas, gas pressure is set to 1.5 mTorr, collision energy for MS/MS fragmentation is optimized to get maximum signal for ion transitions 401 to 257 (for 25OH-D3) and 407 to 263 (for the internal standard).
  • Vitamin D releasing reagent in this example consists of a 50% (weight/volume) solution of l-Butyl-4-methyl pyridinium tetrafluoro borate in water. The mixture is incubated for 20 minutes at room temperature and transferred to autosample of the HPLC system. The following procedure for detection of 25-hydroxyvitamin D 3 is identical to the one given in example 3.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
PCT/EP2007/004924 2006-06-06 2007-06-04 Improved measurement of vitamin d WO2007140962A2 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AT07725793T ATE452343T1 (de) 2006-06-06 2007-06-04 Verbesserte vitamin-d-messung
CA2649338A CA2649338C (en) 2006-06-06 2007-06-04 Improved measurement of vitamin d
EP07725793A EP2030026B1 (de) 2006-06-06 2007-06-04 Verbesserte vitamin-d-messung
JP2009513585A JP4953477B2 (ja) 2006-06-06 2007-06-04 改良されたビタミンｄ測定
DE602007003833T DE602007003833D1 (de) 2006-06-06 2007-06-04 Verbesserte vitamin-d-messung
CN200780021252.0A CN101467048B (zh) 2006-06-06 2007-06-04 改良的维生素d测量方法
US12/326,972 US8003400B2 (en) 2006-06-06 2008-12-03 Measurement of vitamin D

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP06011605 2006-06-06
EP06011605.0 2006-06-06

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/326,972 Continuation US8003400B2 (en) 2006-06-06 2008-12-03 Measurement of vitamin D

Publications (2)

Publication Number Publication Date
WO2007140962A2 true WO2007140962A2 (en) 2007-12-13
WO2007140962A3 WO2007140962A3 (en) 2008-01-31

Family

ID=36646176

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2007/004924 WO2007140962A2 (en) 2006-06-06 2007-06-04 Improved measurement of vitamin d

Country Status (9)

Country Link
US (1) US8003400B2 (de)
EP (1) EP2030026B1 (de)
JP (1) JP4953477B2 (de)
CN (1) CN101467048B (de)
AT (1) ATE452343T1 (de)
CA (1) CA2649338C (de)
DE (1) DE602007003833D1 (de)
ES (1) ES2336382T3 (de)
WO (1) WO2007140962A2 (de)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010100487A1 (en) * 2009-03-02 2010-09-10 Axis-Shield Diagnostics Limited An assay for detecting vitamin d and antibodies therefor
JP2011513710A (ja) * 2008-02-25 2011-04-28 ウオーターズ・テクノロジーズ・コーポレイシヨン サンプル中のビタミンｄ類似体の存在および量の決定のための方法およびキット
WO2011144661A1 (en) * 2010-05-20 2011-11-24 Roche Diagnostics Gmbh Release reagent for vitamin d compounds
WO2012091569A1 (en) 2010-12-28 2012-07-05 Future Diagnostics B.V. Release reagent for vitamin d
CN102692514A (zh) * 2012-06-21 2012-09-26 厦门大学 血液25-羟基维生素d3腺癌检测试剂盒及其制备方法
WO2013072342A1 (en) * 2011-11-18 2013-05-23 Roche Diagnostics Gmbh Release reagent for vitamin d compounds
WO2014122972A1 (ja) 2013-02-06 2014-08-14 富士レビオ株式会社 ビタミンｄの測定方法および測定用キット
CN104076155A (zh) * 2014-07-10 2014-10-01 深圳市新产业生物医学工程股份有限公司 用于检测25-羟基维生素d的检测剂及制备方法和应用
US9244083B2 (en) 2012-11-30 2016-01-26 Siemens Healthcare Diagnostics Inc. Compositions and methods for detecting vitamin D
US9618523B2 (en) 2013-02-28 2017-04-11 Siemens Healthcare Diagnostics Inc. Methods and reagents for determining isomeric analytes
JP2018028547A (ja) * 2008-03-31 2018-02-22 積水化学工業株式会社 液体クロマトグラフィー用部材
US10114030B2 (en) 2014-07-10 2018-10-30 Shenzhen New Industries Biomedical Engineering Co., Ltd Detection agent for detecting 25-hydroxy vitamin D, preparation method and use
US11815517B2 (en) 2011-05-20 2023-11-14 Siemens Healthcare Diagnostics Inc. Antibodies to 25-hydroxyvitamin D2 and D3

Families Citing this family (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7745226B2 (en) 2005-04-06 2010-06-29 Quest Diagnostics Investments Incorporated Methods for detecting vitamin D metabolites
US7972868B2 (en) 2007-11-28 2011-07-05 Quest Diagnostics Investments Incorporated Methods for detecting dihydroxyvitamin D metabolites by mass spectrometry
US8399829B2 (en) 2008-12-18 2013-03-19 Quest Diagnostics Investments Incorporated Vitamin B2 detection by mass spectrometry
US7977117B2 (en) 2009-12-03 2011-07-12 Quest Diagnostics Investments Incorprated Vitamin D metabolite determination utilizing mass spectrometry following derivatization
JP5739903B2 (ja) 2009-12-11 2015-06-24 クエスト ダイアグノスティックス インヴェストメンツ インコーポレイテッド 複合試料中のステロイド化合物の質量分析
US20110240840A1 (en) 2009-12-11 2011-10-06 Quest Diagnostics Investments Incorporated Mass spectrometric determination of cookson-derivatized, non-metabolized vitamin d
JP5351076B2 (ja) * 2010-02-26 2013-11-27 杏林製薬株式会社 オンライン固相抽出による生体試料液中薬剤成分の定量方法
EP2372365A1 (de) * 2010-04-01 2011-10-05 Future Diagnostics B.V. Direkt-Immunoassay auf Vitamin D
US9052308B2 (en) 2011-01-07 2015-06-09 The General Hospital Corporation Assays and methods of treatment relating to vitamin D insufficiency
WO2014179737A2 (en) 2013-05-03 2014-11-06 The General Hospital Corporation Assays and methods of treatment relating to vitamin d insufficiency
WO2012094550A2 (en) * 2011-01-07 2012-07-12 The General Hospital Corporation Assays and methods of treatment relating to vitamin d insufficiency
JP6171333B2 (ja) * 2012-12-26 2017-08-02 東ソー株式会社 安定化したビタミンｄ類標準液
US20140273021A1 (en) * 2013-03-14 2014-09-18 Enzo Biochem, Inc. Vitamin d assays
DE102013205055A1 (de) 2013-03-21 2014-09-25 Orgentec Diagnostika Gmbh Verfahren und Reagenz zur Bestimmung von Vitamin D Metaboliten
DE102013215580A1 (de) * 2013-08-07 2015-02-12 Orgentec Diagnostika Gmbh 25-OH Vitamin D Derivate zur Bestimmung von Vitamin D Metaboliten
CN103467382A (zh) * 2013-09-16 2013-12-25 浙江丽晶化学有限公司 含硫氰酸根离子液体的制备方法及其在聚丙烯腈纺丝加工中的应用
WO2015061598A1 (en) 2013-10-24 2015-04-30 Quidel Corporation Systems and methods for whole blood assays
CN105372337B (zh) * 2014-08-07 2017-11-10 山东达因海洋生物制药股份有限公司 一种检测维生素d滴剂中维生素d含量的方法
WO2017039574A1 (en) * 2015-08-28 2017-03-09 Enzo Life Sciences, Inc. Vitamin d assays
US10591445B2 (en) 2015-11-25 2020-03-17 Del Mar Pharmaceuticals (Bc) Ltd. Methods for analysis and resolution of preparations of dianhydrogalactitol and derivatives or analogs thereof
WO2017100457A1 (en) * 2015-12-11 2017-06-15 Opko Diagnostics, Llc Fluidic systems involving incubation samples and/or reagents
CN106199015B (zh) * 2016-06-27 2018-08-17 江苏拜明生物技术有限公司 用于血清样品检测的维生素d释放剂、制备方法及应用
CN108982882A (zh) * 2018-06-22 2018-12-11 卢氏实验室公司 一种数字化信息化维生素d快速检测系统和方法
CN110794065A (zh) * 2018-08-03 2020-02-14 郑州泰丰制药有限公司 利用双三元检测骨化三醇软胶囊含量的方法
CN108663461B (zh) * 2018-08-06 2021-03-19 通标标准技术服务(上海)有限公司 一种测定奶粉中维生素d的方法
CN109358148A (zh) * 2018-09-20 2019-02-19 杭州和合医学检验实验室有限公司 血液中维生素d的检验方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999067211A1 (de) * 1998-06-25 1999-12-29 Immundiagnostik Gesellschaft Für Produktion Und Vertrieb Von Labordiagnostika Mbh FUNKTIONELLE VITAMIN-D-DERIVATE UND VERFAHREN ZUR BESTIMMUNG VON 25-HYDROXY- UND 1α,25-DIHYDROXY-VITAMIN-D
WO2002046746A2 (en) * 2000-12-06 2002-06-13 Immunodiagnostic Systems Ltd Method for detection of vitamin d metabolites
WO2002057797A2 (en) * 2001-01-16 2002-07-25 Quest Diagnostics Inc Vitamin d assay

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5821020A (en) * 1995-07-14 1998-10-13 Nhh Biologics Vitamin D assay
AU1429397A (en) * 1995-12-29 1997-07-28 A And D Assay, Incorporated Labeled vitamin d compounds and the use thereof
US20040132104A1 (en) * 2003-01-07 2004-07-08 Sackrison James L. Vitamin D assay
ATE530915T1 (de) * 2005-09-29 2011-11-15 Hoffmann La Roche Freisetzungsreagens für vitamin-d-verbindungen

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999067211A1 (de) * 1998-06-25 1999-12-29 Immundiagnostik Gesellschaft Für Produktion Und Vertrieb Von Labordiagnostika Mbh FUNKTIONELLE VITAMIN-D-DERIVATE UND VERFAHREN ZUR BESTIMMUNG VON 25-HYDROXY- UND 1α,25-DIHYDROXY-VITAMIN-D
WO2002046746A2 (en) * 2000-12-06 2002-06-13 Immunodiagnostic Systems Ltd Method for detection of vitamin d metabolites
WO2002057797A2 (en) * 2001-01-16 2002-07-25 Quest Diagnostics Inc Vitamin d assay

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HOLMBERG I ET AL: "DETERMINATION OF 25-HYDROXY VITAMIN D-3 IN SERUM BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AND ISOTOPE DILUTION-MASS SPECTROMETRY" SCANDINAVIAN JOURNAL OF CLINICAL AND LABORATORY INVESTIGATION, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 44, no. 4, 1984, pages 275-282, XP009052947 ISSN: 0085-591X *
SHIMADA K ET AL: "Gas chromatography and high-performance liquid chromatography of natural steroids" JOURNAL OF CHROMATOGRAPHY A, ELSEVIER, AMSTERDAM, NL, vol. 935, no. 1-2, 23 November 2001 (2001-11-23), pages 141-172, XP004322069 ISSN: 0021-9673 *
ZERWEKH J E: "THE MEASUREMENT OF VITAMIN D: ANALYTICAL ASPECTS" ANNALS OF CLINICAL BIOCHEMISTRY, BRITISH MEDICAL ASSOCIATION, LONDON, GB, vol. 41, no. PART 4, July 2004 (2004-07), pages 272-281, XP008056470 ISSN: 0004-5632 cited in the application *

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011513710A (ja) * 2008-02-25 2011-04-28 ウオーターズ・テクノロジーズ・コーポレイシヨン サンプル中のビタミンｄ類似体の存在および量の決定のための方法およびキット
JP2018028547A (ja) * 2008-03-31 2018-02-22 積水化学工業株式会社 液体クロマトグラフィー用部材
JP2019032346A (ja) * 2008-03-31 2019-02-28 積水化学工業株式会社 液体クロマトグラフィー用部材
WO2010100487A1 (en) * 2009-03-02 2010-09-10 Axis-Shield Diagnostics Limited An assay for detecting vitamin d and antibodies therefor
US10451639B2 (en) 2010-05-20 2019-10-22 Roche Diagnostics Operations, Inc. Release reagent for vitamin D compounds
CN102906570A (zh) * 2010-05-20 2013-01-30 霍夫曼-拉罗奇有限公司 维生素d化合物的释放制剂
WO2011144661A1 (en) * 2010-05-20 2011-11-24 Roche Diagnostics Gmbh Release reagent for vitamin d compounds
KR101494952B1 (ko) * 2010-05-20 2015-02-23 에프. 호프만-라 로슈 아게 비타민 d 화합물용 방출 시약
EP3327442A1 (de) * 2010-05-20 2018-05-30 F. Hoffmann-La Roche AG Freisetzungsreagens für vitamin-d-verbindungen
JP2014501392A (ja) * 2010-12-28 2014-01-20 フューチャー・ダイアグノスティックス・ベーフェー ビタミンｄのための放出試薬
US10422805B2 (en) 2010-12-28 2019-09-24 Future Diagnostics B.V. Release reagent for vitamin D
WO2012091569A1 (en) 2010-12-28 2012-07-05 Future Diagnostics B.V. Release reagent for vitamin d
US11815517B2 (en) 2011-05-20 2023-11-14 Siemens Healthcare Diagnostics Inc. Antibodies to 25-hydroxyvitamin D2 and D3
EP3467510A1 (de) * 2011-11-18 2019-04-10 F. Hoffmann-La Roche AG Freisetzungsreagens für vitamin-d-verbindungen
CN103946710A (zh) * 2011-11-18 2014-07-23 霍夫曼-拉罗奇有限公司 维生素d化合物的释放制剂
US12013405B2 (en) 2011-11-18 2024-06-18 Roche Diagnostics Operations, Inc. Release reagent for vitamin D compounds
AU2012338908B2 (en) * 2011-11-18 2018-03-22 F. Hoffmann-La Roche Ag Release reagent for vitamin D compounds
US11187709B2 (en) 2011-11-18 2021-11-30 Roche Diagnostics Operations, Inc. Release reagent for vitamin D compounds
WO2013072342A1 (en) * 2011-11-18 2013-05-23 Roche Diagnostics Gmbh Release reagent for vitamin d compounds
CN102692514A (zh) * 2012-06-21 2012-09-26 厦门大学 血液25-羟基维生素d3腺癌检测试剂盒及其制备方法
US9244083B2 (en) 2012-11-30 2016-01-26 Siemens Healthcare Diagnostics Inc. Compositions and methods for detecting vitamin D
US10203344B2 (en) 2012-11-30 2019-02-12 Siemens Healthcare Diagnostics Inc. Compositions and methods for detecting vitamin D
WO2014122972A1 (ja) 2013-02-06 2014-08-14 富士レビオ株式会社 ビタミンｄの測定方法および測定用キット
US10571479B2 (en) 2013-02-06 2020-02-25 Fujirebio Inc. Vitamin D measurement method and measurement kit
US9618523B2 (en) 2013-02-28 2017-04-11 Siemens Healthcare Diagnostics Inc. Methods and reagents for determining isomeric analytes
CN104076155A (zh) * 2014-07-10 2014-10-01 深圳市新产业生物医学工程股份有限公司 用于检测25-羟基维生素d的检测剂及制备方法和应用
US10114030B2 (en) 2014-07-10 2018-10-30 Shenzhen New Industries Biomedical Engineering Co., Ltd Detection agent for detecting 25-hydroxy vitamin D, preparation method and use
CN104076155B (zh) * 2014-07-10 2016-08-31 深圳市新产业生物医学工程股份有限公司 用于检测25-羟基维生素d的检测剂及制备方法和应用

Also Published As

Publication number Publication date
ATE452343T1 (de) 2010-01-15
WO2007140962A3 (en) 2008-01-31
EP2030026B1 (de) 2009-12-16
CA2649338A1 (en) 2007-12-13
CN101467048A (zh) 2009-06-24
JP4953477B2 (ja) 2012-06-13
CN101467048B (zh) 2014-04-02
ES2336382T3 (es) 2010-04-12
DE602007003833D1 (de) 2010-01-28
EP2030026A2 (de) 2009-03-04
JP2009540275A (ja) 2009-11-19
US20100285603A1 (en) 2010-11-11
CA2649338C (en) 2011-07-12
US8003400B2 (en) 2011-08-23

Similar Documents

Publication Publication Date Title
CA2649338C (en) Improved measurement of vitamin d
JP7157212B2 (ja) 質量分析によってジヒドロキシビタミンｄ代謝物を検出するための方法
US11808773B2 (en) Mass spectrometry of steroidal compounds in multiplexed patient samples
US11885820B2 (en) Vitamin D metabolite determination utilizing mass spectrometry following derivatization
WO2011072168A1 (en) Mass spectrometric determination of cookson-derivatized, non-metabolized vitamin d

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200780021252.0

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07725793

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 2649338

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2009513585

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2007725793

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: RU